ABSTRACT

Pfs expression is required for several metabolic pathways and limits the production of autoinducer-2, a molecule proposed to play a central role in interspecies quorum sensing. The present study reveals physiological conditions and promoter DNA elements that regulate Escherichia coli pfs transcription. Pfs transcription is shown to rely on both sigma 70 and sigma 38 (rpoS), and the latter is subject to induction that increases pfs expression. Transcription is maximal as the cells approach stationary phase, and this level can be increased by salt stress through induction of sigma 38-dependent expression. The pfs promoter is shown to contain both positive and negative elements, which can be used by both forms of RNA polymerase. The negative element is contained within the overlapping dgt promoter, which is involved in purine metabolism. Consideration of the physiological roles of sigma 38 and dgt leads to a model for how autoinducer production is controlled under changing physiological conditions.

The pfs gene, encoding the 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase enzyme, is required for polyamine synthesis, adenine and methionine salvage, removal of toxic metabolites, and creation of luxS-dependent autoinducers (AIs) of quorum sensing in Escherichia coli and related bacteria (5, 22, 26). As quorum sensing involves cell-to-cell signaling, it would be expected to be a highly regulated process. Gene fusions have shown that the pfs-luxS pathway of AI-2 synthesis is limited by the expression of pfs rather than luxS (3). Pfs expression rises as cells approach stationary phase and then falls. AI-2 synthesis follows a similar pattern (31). AI-2 is excreted until stationary phase is reached, when it begins to be imported (27, 31). The sources of these regulatory events are not known, and their significance with respect to quorum sensing is not well understood. In interspecies quorum sensing, it has been proposed that products of the pfs/luxS pathway, AI-2 molecules, accumulate from multiple bacterial species and then signal the population to behave coordinately (12, 32). The issue is important because quorum sensing is a widespread phenomenon and has been invoked as a potential target of therapeutic intervention (2).

Because the expression of pfs rises as stationary phase approaches (3), we considered the possibility that the source of the rise might be induction of rpoS-dependent transcription. rpoS encodes sigma 38, which is induced in response to multiple stresses and directs the transcription of numerous genes that deal with physiological stress (10). Its initial expression pattern mimics that seen for pfs; sigma 38 is induced as cells sense the stresses that will lead to the halt in growth that is characteristic of stationary phase (14). If sigma 38 were the source of pfs regulation, this would have implications for events such as quorum sensing, which is proposed to be controlled by the AI system.

We now report that rpoS-dependent expression is indeed the source of pfs induction. Overall, pfs transcription is shown to be the product of both sigma 70- and sigma 38-dependent transcription. The DNA elements responsible for pfs transcription have been analyzed and include a standard positive element and a negative element apparently related to transcription of the divergent overlapping dgt promoter involved in nucleotide breakdown. Consideration of these data in the context of pfs metabolism leads to a proposal for the kinds of physiological conditions expected to trigger induction of AI-2 and helps to evaluate the role of this pathway in interspecies quorum sensing and metabolism.

MATERIALS AND METHODS

Bacterial strains and growth conditions.E. coli K-12 (ATCC 10798) or RJ4099 (CAG4000 proP-104::TnphoA′-4 katF13::Tn10; lacks the σ38 gene and carries the lacZ gene under the control of a σ38 promoter, proP [which lacks sigma 38]) cells were grown overnight in 5 ml of Luria broth at 37°C. The overnight culture were diluted 1:200 in Luria broth plus 0.5% glucose at 37°C, and samples were taken periodically during growth from the exponential phase into the early stationary growth phase. For salt stress, the culture was divided at an optical density at 600 nm (OD600) of 0.2, and one received 5 M NaCl to make a final added concentration of 0.4 M NaCl. At subsequent times, samples were harvested, quickly centrifuged, frozen, and stored at −70°C for RNA extraction. The data for analysis were taken before stress and at the transition to stationary phase at an OD600 of 1.2 in each case. The salt-stressed cells were delayed in growth. RJ4099 is defective in the production of several mRNAs, including pfs, otsB, and osmY, and addition of an rpoS expression vector complements this defect (unpublished data).

Plasmids.Serial upstream pfs promoter truncations were constructed by ligation of cloned promoter fragments with approximate upstream endpoints at −55, −40, −30, and −20 and then in 5-bp intervals through the RNA polymerase binding site. The segments were cloned from the E. coli chromosome by PCR and inserted between the BamHI and HindIII sites of plasmid pTH8. The parent −55 was obtained by using a 228-bp fragment starting 87 bp upstream of the +1 amino acid start of the pfs gene. All clones were inserted so that transcription would end at a common termination site on the plasmid and were studied in E. coli K-12 cells.

Primers and sequencing.A sequencing kit (USB) was used according to the manufacturer's instructions. One hundred twenty nanograms of plasmid in 20-μl reaction mixtures was cycled 25 times for 3.5 min at 94°C, 60°C for 30 s, and 72°C for 1 min. The pfs primer was 5′-GACGGTTTTCGATTTTGTCACGCAGCAGCG-3′, and the dgt primer was 5′-CCGCAGGATCTCATGTTCGGTTTTAACGCC-3′.

RNA analysis.RNA was extracted with the RNeasy kit protocol (QIAGEN), and total RNA was assayed spectrophotometrically as specified. Specific mRNA was detected by extension of designed downstream primers. Ten microliters of reaction mixture for primer extension analysis contained 10 μg of total RNA, 10 nM radiolabeled primer, reverse transcriptase buffer (Promega), and 0.5 mM deoxynucleoside triphosphates. Samples were initially incubated at 85°C for 3 min. The reaction used 5 U of reverse transcriptase at 45°C for 10 min and then 1 h 10 min at 37°C. Urea stop dye was added, and the samples were loaded and run on a 6% polyacrylamide gel at 21 W. Radioactive bands were visualized and quantified by phosphorimager analysis. RNA samples were prepared at least three separate times, and the average was taken.

RESULTS

Pfs transcription is transiently induced via sigma 38.Prior experiments using Salmonella with lacZ-pfs promoter fusions showed that pfs expression rises as stationary phase is approached and then falls as the cells enter stationary phase (3). We conducted similar experiments with E. coli but with a direct primer extension assay for pfs mRNA. Figure 1A, light bars, shows that pfs mRNA increases during exponential phase and reaches a peak at an OD600 of 1.2, in accord with the fusion results. Figure 1B, light bars, shows the subsequent decline as the cells enter stationary phase, also in agreement with prior data from fusions.

sigma 38 induces pfs transcription. Primer extension was used to measure pfs mRNA from wild-type K-12 (light bars) or RJ4099 (dark bars). (A) The experiment was done as the cells approached stationary phase, with RNA harvested at the indicated cell densities. The inset shows primer extension products with the indicated ODs and strains. (B) The experiment shows data taken over a 1-h time course in stationary phase. In panel B, data for each strain are independently normalized to the starting level.

To assess the potential role of sigma 38 in this regulation, the experiments were repeated with strain RJ4099 (33), which is deficient in sigma 38 production. Sigma 38 protein is known to exist at very low levels early in exponential phase and accumulate as the cells approach stationary phase (13, 14). The pfs transcription data show that RJ4099 cells fail to induce pfs transcription as stationary phase is approached (Fig. 1A, dark bars). This indicates that the pfs induction requires sigma 38 RNA polymerase.

Sigma 38 levels are known to continue rising as the cells enter stationary phase (13, 14). Nonetheless, pfs transcription declines during this time and this occurs in both the wild-type K-12 and sigma 38-deficient RJ4099 strains (Fig. 1B). There is no indication of retained induction by sigma 38, as both strains appear to produce equivalent amounts of mRNA. Thus, the induction of sigma 38-dependent pfs transcription is a transient event.

Sigma 38 is induced by a variety of stresses, and the extent to which sigma 38-dependent transcription is increased depends on the cumulative effect of the stressors (10). We tested the extent of induction of pfs transcription after exposing the cells to one of these stresses, hyperosmotic shock. This stress is common to the human gastrointestinal tract (6, 21) and other environments and leads to the production of elevated amounts of sigma 38. Some sigma 38-dependent genes (28, 29) are strongly induced under these conditions, but most are weakly induced. The salt shock leads to a delay in growth, which later resumes as the strongly induced genes allow adaptation to the high-salt conditions (9, 16).

Cells were challenged with added 400 mM NaCl in early exponential phase (OD600 = 0.2) and assayed at subsequent times for pfs mRNA. The experiments were done with both the wild-type K-12 (light bars in Fig. 2) and sigma 38-deficient RJ4099 (dark bars) strains. Figure 2 presents the results just prior to challenge (leftmost pair of bars, an OD of 0.2) and at the transition into stationary phase (rightmost pair of bars, NaCl at an OD of 1.2). Comparison of the leftmost and rightmost light bars shows that the level of pfs RNA has approximately doubled in the salt-challenged cells. All of this pfs mRNA induction is due to the presence of sigma 38, as pfs transcription in RJ4099 cells remains unchanged (dark bars). Comparison with the data from unchallenged cells at the transition to stationary phase (middle pair of bars) indicates that the induction is partly due to the cells approaching stationary phase, as described above and partly due to the salt challenge. This was quantified by averaging numerous experiments; the results indicate that about half of the induction is due to the approach to stationary phase and about half is due to the salt challenge.

Salt stress induces pfs transcription via sigma 38. mRNA was assayed (top) prior to addition of 0.4 M NaCl at an OD600 of 0.2 and later at an OD600 of 1.2. The strains used were wild-type K-12 (light bars) and RJ4099 (dark bars). The data are quantified below with calculated error bars from multiple experiments. The OD600 of 1.2 was reached approximately 80 min later in the case of salt-stressed cells.

We conclude that under the conditions of this salt challenge, pfs transcription can double because of the presence of sigma 38. In a natural environment, the induction could be higher or lower, depending on what stressors are present and how they combine to increase the level of sigma 38 protein. The timing of the spike in pfs transcription might also depend on this mixture of stressors. These considerations are important, as they outline the types of conditions expected to induce pfs transcription, which limits the production of AIs.

Elements regulating pfs transcription.The elements controlling pfs transcription are unknown. At most promoters, the RNA polymerase initially binds roughly between positions −50 and +20 (8). To learn if the DNA element(s) responsible for the changes in transcription observed here lie within these limits, we cloned the pfs promoter on a plasmid. The upstream boundary was near position −55, as judged by the location of the pfs start site in primer extension studies (see mapping below). Plasmid-based pfs promoter-directed transcription in vivo was assayed by use of a primer that hybridizes to a region of the plasmid downstream from the inserted pfs promoter. pfs mRNA was measured under the same growth conditions as in the chromosome-based experiments just described.

Figure 3A shows that the pattern of changes in pfs transcription during growth is unchanged in the context of this plasmid. The quantitative extent of inhibition as the cells approach stationary phase appears to be similar on the plasmid and the chromosome (compare to Fig. 1A). The induction upon approach to stationary phase also occurs, although it is not clear whether it is the same as or less than in the chromosomal context. The salt challenge induction also appears to be largely intact in the plasmid context (Fig. 3B). We conclude that the elements regulating pfs transcription are located within the DNA sequences retained on this plasmid.

Regulation is largely retained on a plasmid lacking far upstream sequences. The pfs promoter was cloned from positions −55 to +20 (see mapping below), and mRNA was assayed by primer extension at the indicated ODs. The primer extension products are shown in the insets, and the bars represent their levels. Panel B shows the result of adding 0.4 M NaCl.

To locate the DNA sequence element(s) responsible for determining the extent of transcription, a series of promoter truncations were constructed and assayed. These progressively remove DNA sequences beginning upstream and then entering the region that typically contains the core promoter elements that direct basal transcription. Plasmid-based pfs transcription in vivo was assayed for each of these, and the results are presented in Fig. 4.

The pfs promoter contains positive and negative elements. The series of upstream deletions were made and assayed for pfs mRNA levels at an OD of approximately 1.0. The data are shown for RJ4099 cells, and data from K-12 cells show the same trend. Plasmid (underlined) and pfs DNAs are shown below.

Two aspects of these data are most notable. First, removal of DNA sequences from parent clone −55 to form clone −15 results in a significant increase in transcription. This implies that an inhibitory element lies between these limits (new promoter elements are not brought in [see below]). Second, removal of the 5 bp downstream from the −15 clone to form clone −10 results in nearly complete loss of transcription, implying that a positive element lies between these limits. The locations of these elements were not different for experiments with the K-12 and RJ4099 strains (data not shown). The results identify both positive and negative elements for pfs transcription.

Properties of regulatory elements.In principle, the expression of pfs mRNA could be done directly by sigma 38 RNA polymerase or be an indirect consequence of a regulator transcribed via sigma 38. A hypothetical required regulator would need to act downstream of position −55, as the plasmid just described retains regulation. No inverted repeat sequences that signify regulatory protein binding are present, and potential regulators of pfs transcription have not been proposed. To assess the ability of sigma 38 RNA polymerase to directly transcribe pfs, experiments were done with a purified system. This transcription system contains core RNA polymerase and purified sigma factors as the only sources of proteins. The same series of promoter deletions were tested in this purified system to learn if the pattern of sigma 38 transcription relies on the same elements used in vivo.

Figure 5 shows a panel of RNA products of purified sigma 38 RNA polymerase transcription with the promoter deletion series (quantified with dark bars). As deletions enter from the upstream region, transcription decreases slightly until clone −15, whereupon it increases. The next deletion (−10) essentially eliminates transcription. This is the same pattern observed when transcription in vivo was assayed (Fig. 4). We conclude that purified sigma 38 RNA polymerase, in the absence of added regulators, can use the same promoter elements that are used in vivo.

In vitro transcription with purified sigma 38 RNA polymerase. The same constructs assayed in vivo in Fig. 4 were assayed here in vitro. The only macromolecules present were RNA polymerase holoenzyme and plasmid DNA. The autoradiograph shows the products of sigma 38 transcription. The dark bars quantify the results and compare them to levels of RNA obtained when sigma 70 was substituted for sigma 38 (light bars). For each holoenzyme, the levels were normalized to that of construct −55. The start sites were at similar locations for sigma 38 transcription and sigma 70 transcription.

As sigma 38 promoter elements strongly overlap those used by sigma 70, promoters are often transcribed by both forms of RNA polymerase (11). The above in vivo data for pfs transcription are consistent with this view. To evaluate this possibility, the experiments were repeated with purified sigma 70 RNA polymerase. The data for sigma 70 (light bars) are collected in Fig. 5 along with transcription from purified sigma 38 RNA polymerase (dark bars). The comparisons show that sigma 38 and sigma 70 transcription relies on the same positive and negative elements, which match those used in vivo. We infer that the in vivo regulatory pattern of pfs transcription can be reproduced in vitro with either form of RNA polymerase in the absence of other regulators.

The positive regulatory element is defined by the drastic loss of transcription when progressing from clone −15 to clone −10. To evaluate the role of the sequences within this segment, the pfs start site (which is the same for both RNA polymerases, as is commonly observed [11]) was mapped (Fig. 6). The +1 location is preceded by a segment with a strong resemblance to a known positive consensus element for RNA polymerase recognition in the appropriate location. This pfs sequence (TGGTAAACT) is boxed in Fig. 6 and matches the established “extended −10” consensus sequence (TGxTATAAT) at six of eight positions (19). Such elements have been best studied in the context of sigma 70 transcription. sigma 38 transcription and sigma 70 transcription are both known to use an element in this location, with the sigma 38 sequence specificity appearing to be much lower; it is common for a single element to be used by both forms of RNA polymerase (11). The deletion to form clone −10 would remove the upstream TG dinucleotide, which is essential for transcription in the absence of a −35 element, consistent with the observed loss of pfs transcription. We suggest that the positive element for pfs transcription is simply related to the use of this sequence by both forms of RNA polymerase.

The negative element is embedded within the divergent, overlapping dgt promoter. pfs (left) and dgt (right) primers were used to map the 5′ ends of mRNAs. In vivo mRNA extension products were run in parallel with in vitro-generated sequencing markers with the same primers. The arrow and arrowheads point to the deduced transcription start sites. The DNA sequence of the region is shown below the autoradiograph. Transcription start sites of the overlapping promoters and the endpoints of selected upstream plasmid deletions used in Fig. 4 are indicated. The proposed pfs extended −10 sequence is boxed and compared to the consensus. The −35 region in clone −15 is boxed with dashes and does not match the known consensus.

The negative element is defined by the deletion of sequences just upstream from this location, corresponding to the gain in transcription when progressing from clone −20 to clone −15. This gain does not appear to be caused by the introduction of a new promoter from plasmid sequences; all of the clones contain the same upstream promoter sequences, and all preserve the wild-type pfs transcription start site. Nor is there any indication that a new −35 element has been juxtaposed, as there is no properly located sequence closely resembling the established −35 consensus (TTGACA), which can be used by sigma 38 or sigma 70 (7). At the required location of such a −35 element only, there is a two-of-six match to the consensus (dashed box in Fig. 6), similar to the expectation from a random match.

These considerations indicate that the increase in transcription with clone −15 is not due to artifactual juxtaposition of a new −35 consensus element and must have some other source. We note that the pfs gene is immediately adjacent to the facing dgt gene, encoding a dGTP phosphatase (30). Thus, it is possible that the −20-to-−15 deletion removes DNA needed by RNA polymerase at the hypothetical dgt promoter, thereby favoring use of the pfs promoter and increasing pfs transcription.

To test the possibility that the pfs and dgt promoters overlap, we attempted to locate the start site of dgt transcription. Only low levels of transcription were detected, but this allowed mapping of two mRNA start sites (Fig. 6, right). The results demonstrate that the pfs and dgt promoters overlap, as the RNA polymerases would have between 35 and 55 bp in common in the expected approximately 70-bp binding sites. Taken together, the data show that pfs transcription increases when one removes DNA sequences that lie within the dgt promoter immediately downstream of a dgt transcription start site. Overlapping promoter arrangements such as that indicated here are known to be associated with “promoter interference” of the type suggested by these pfs and dgt data (1, 23).

Overall, the data indicate that the use of pfs transcription elements in vivo can be reproduced in a system containing no regulators and suggest that a standard −10 element and an overlapping dgt promoter contribute to the positive and negative regulation observed both in vitro and in vivo.

DISCUSSION

These results have defined physiological conditions and DNA elements that contribute to determining the level of pfs transcription. Pfs (or the 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase enzyme) determines the flow of metabolites through several pathways and limits production of the luxS-dependent AI molecules that are proposed to be involved in interspecies quorum sensing (18, 26). As a signaling pathway, quorum sensing would be subject to regulation, and the present results bear on that issue.

Two aspects of the new data are particularly relevant to understanding what causes levels of pfs mRNA to change. First, the results identify sigma 38, the product of the rpoS gene, to be required for the known induction (3) as the cells approach stationary phase. They further show that salt stress, known to lead to sigma 38 accumulation (14), can modestly induce pfs transcription. A positive transcription element is identified that likely mediates these induction events by directing sigma 38 transcription as sigma 38 accumulates at the onset of stationary phase (14). This element is shown to function to direct transcription by sigma 38 RNA polymerase in a purified system. The main implication of these observations is that the potential for pfs transcription, and thus the potential for AI synthesis, will increase with conditions that induce the general stress factor sigma 38.

Another clue to pfs regulation comes from data showing that deletion of a short DNA segment leads to increased transcription. This phenomenon was reproduced in a highly purified system containing either the sigma 38 or the sigma 70 form of RNA polymerase, ruling out the use of this element as a binding site for an unknown regulatory protein. The deleted DNA corresponds to the region immediately downstream from the observed start site for transcription of the dgt gene; the dgt and pfs promoters are arranged face to face and are expected to overlap by up to 50 bp. Overlapping promoter arrangements often lead to coordinated expression as properties of RNA polymerase bound to one promoter (dgt, for example) can influence the ability of RNA polymerase to transcribe the other (pfs for example) (23). Thus, to understand pfs regulation, one needs to explore the physiological role of dgt.

Dgt is involved in purine breakdown, catalyzing the removal of phosphates from dGTP (30). Stationary-phase cells excrete purine breakdown products into the medium (20), and both pfs and dgt contribute to purine breakdown (Fig. 7). pfs is central to this process, as both characterized pfs enzymatic reactions produce adenine (from either S-adenosylhomocysteine or methylthioadenosine), which is recycled during exponential growth (not shown in Fig. 7) and broken down and detected in the medium of stationary-phase cells (4, 20, 25, 26). Thus, pfs and dgt work together to break down purine under conditions of purine excess. In batch culture, purine excess results from the imbalance between synthesis and consumption that occurs as cells approach stationary phase, when purine consumption by macromolecular synthesis abruptly slows. We note that this known production of excess purine is coincident with the observed maximal induction of pfs transcription. As synthesis of new purine is feedback regulated, the excess would be transient and the need for pfs would decline with the falling purine pools; the subsequent decline is also observed for pfs transcription. Thus, both the rise in pfs transcription that occurs as the cells approach stationary phase and the subsequent decline are consistent with a role for pfs in purine metabolism.

These considerations suggest that cells have evolved a mechanism for inducing pfs transcription when it is needed to deal with the excess purine that accumulates transiently at the onset of stationary phase. sigma 38 is naturally induced as cells approach stationary phase (14), and the present data show that this is responsible for pfs induction at this time. The products of pfs metabolism (Fig. 7) would be excreted as adenine breakdown products along with their pathway partners, the luxS-dependent AI molecules, which are also known to be excreted at this time (12). It is not known how pfs transcription is progressively down-regulated after the transition to stationary phase, when sigma 38 levels are high, but such down-regulation is not unusual and may be global rather than gene specific (24).

In this view, AIs should be produced in large amounts when E. coli and related bacteria need to excrete purine. The key question then becomes the following: have cells taken advantage of this to use the pathway “by-products,” AIs, to signal quorum sensing? Finding the answer to this question will rely on a better understanding of the physiological conditions that induce processes that rely on quorum sensing.